Glutathionylspermidine synthetase and processes for recovery and use thereof

ABSTRACT

The present invention describes an enzyme showing glutathionylspermidine synthetase-activity and being distinct from known enzymes with similar activities in several physicochemical parameters, a novel process to isolate said enzyme from  Crithidia fasciculata,  tools for the production thereof in genetically transformed organisms, and its use as a molecular target for the discovery of trypanocidal drugs.

[0001] This is a continuation of International Application No.PCT/EP97/06982 filed Dec. 12, 1997, the entire disclosure of which isincorporated herein by reference.

[0002] Glutathionylspermidine synthetase (GspS) catalyzes the first oftwo steps of trypanothione biosynthesis, the synthesis ofglutathionylspermidine (Gsp) from glutathione (GSH) and spermidine withthe consumption of ATP (1). Trypanothione (N¹,N⁸-bis(glutathionyl)spermidine, TSH) is a metabolite unique totrypanosomatids such as Trypanosoma species, Leishmania species, andCrithidia fasciculata (2). These parasites comprise pathogens causingwidespread and difficult-to-treat tropical diseases such as Africansleeping sickness (T. brucei gambiense or T. brucei rhodesiense), Chagasdisease (T. cruzi), kala azar (L. donovani), oriental sore (L. tropica)and mucocutaneous leishmaniasis (L. braziliensis). Others (e.g. T.congolense) affect domestic animals, whereas C. fasciculata ispathogenic to insects only.

[0003] Since the discovery of TSH in 1985 (3, 4), the pathways for itssynthesis and utilization have attracted considerable interest aspotential targets for selective therapeutic intervention (5, 6). In alltrypanosomatids TSH substitutes for GSH in the defense againsthydroperoxides and derived reactive oxygen species because of itsability to reduce peroxides either enzymatically (7-9) or spontaneously(10). It thereby protects the parasitic trypanosomatids, whichapparently are deficient in catalase and glutathione peroxidases (11),against oxidative stress for instance during host-defense reactions (9,12, 13). Trypanothione disulfide thereby formed is reduced by theNADPH-dependent trypanothione reductase (14, 15), a flavoproteinhomologous to glutathione reductase which together with glutathioneperoxidases (16, 17) constitutes a major part of the defense system ofthe host (18, 19). The precursor of TSH, Gsp, may have a distinctbiological role. It was first identified in Escherichia coli (20), whereit remains unprocessed to TSH due to the apparent lack of TSHsynthetase. In E. coli GspS, and consequently Gsp, is prominent in thestationary phase (20, 21). Similarly, in C. fasciculata Gsp increasessubstantially during the transition of growth phase to stationary phase,while TSH simultaneously drops (22). These fluctuations of GSHconjugates or the associated variations in cellular spermidine levelshave tentatively been implicated in growth regulation (2, 20, 21). Themajor biological function of TSH in trypanosomatids is to serve as areducing substrate for thioredoxin-like proteins called tryparedoxins(23). Tryparedoxins in turn may have a variety of functions in replacingthioredoxin which so far could never be identified in trypanosomatids. Aprominent role of tryparedoxin consists in the regeneration oftryparedoxin peroxidase after reaction thereof with a hydroperoxide suchas H₂O₂, a fatty acid hydroperoxide, or a hydroperoxide of a completelipid (24). Thereby, GspS together with trypanothione synthetase,trypanothione reductase, tryparedoxin, and tryparedoxin peroxidaseconstitutes the most complex system to protect trypanosomatids againstoxidative damage. By analogy, reduced tryparedoxin may also substituefor thioredoxin in other pathways, e.g. in the reduction ofribonucleotides thereby becoming essential for the entire nucleic acidmetabolism in trypanosomatids.

[0004] The first enzyme catalyzing synthesis of Gsp has once beenisolated in trace amounts from C. fasciculata (0.5 mg from 500 g wetcell mass) and characterized in terms of the apparent MW, kineticparameters, and substrate specificity (1).

[0005] Another deduced amino acid sequence obtained from C. fasciculatafirst claimed to represent a trypanothione-synthetase-like protein(acc.number U66520) was submitted to Genbank on Aug. 9, 1996, becameavailable to the public in February 1997 and was reported to be thesequence of glutathionylspermidine synthetase without, however,providing any experimental data supporting this assignment. In terms ofsize and sequence, this putative GspS of C. fasciculata is not identicalwith the GspS of C. fasciculata described in the present invention. Thisprotein differs substantially from GspS described here in molecular mass(90 (1) versus 78-79 kDa, respectively) and pH optimum (6.5 (1) versus7.5). Also, the previously described enzyme reportedly hydrolyzed ATP inthe absence of spermidine (1), whereas such activity was not detectablein GspS as characterized here. Taken together, these discrepanciesdemonstrate that the two preparations can not be considered identical orequivalent.

[0006] An enzyme catalyzing the analogous reaction in E. coli hasrecently been cloned. Surprisingly, this GspS also exhibits asubstantial amidase activity with Gsp as substrate. The simultaneouscatalysis of Gsp synthesis and breakdown results in an apparently futileATP consumption, the biological role of which remains speculative (25,26). Since E. coli does not produce TSH, its GspS has obviously to beseen in a biological context distinct from trypanosomal TSH metabolism,and also the structural and phylogenetic relationship of bacterial andtrypanosomal GspS remains to be investigated.

[0007] Most importantly, we here describe a method for purification ofGspS from C. fasciculata yielding an enzyme pur enough to be sequenced.The partial amino acid sequence enables the identification of thepertinent gene and the heterologous expression therof by methods knownper se, thereby making GspS available for the identification of specificinhibitors useful as trypanocidal drugs. Further, we have invented asimple and convenient method to partially purify GspS from C.fasciculata. Such preparation does not catalyze any ATP hydrolysis inthe absence of further GspS substrates and cofactors, i. e. spermidineand magnesium. This implies that GspS activity and inhibition thereofcan be specifically measured simply by liberation of inorganic phosphatefrom ATP in such partially purified GspS. This test system is easilyautomatized for large scale inhibitor screening.

[0008] Thus, one embodiment of the invention concerns a proteincharacterized by its ability to catalyze the synthesis ofglutathionylspermidine with a pH optimum of about 7.5.

[0009] The protein according to the invention is further characterizedby an apparent molecular weight of 78,000±3000 Da.

[0010] The protein is further characterized by comprising partialsequences shown in FIG. 1 and being homologous to glutathionylspermidinesynthetase/amidase of Escherichia coli. The protein according to theinvention is further characterized by being isolated from a species ofthe family trypanosomatidae or produced in any other species byrecombinant DNA techniques making use of the partial amino acidsequences shown in FIG. 1, genetic probes or primers derived thereof orencoding nucleic acid sequences thus obtained, e.g. SEQ ID NO1 (FIG. 2)or any useful part thereof.

[0011] The protein according to the invention is further characterizedby comprising the partial amino acid sequence deduced from the nucleicacid sequence SEQ ID NO1.

[0012] The protein according to the invention is further characterizedby comprising or having a sequence which is at least 70%, preferntially75% identical to that deduced from SEQ ID NO1, respectively.

[0013] The protein according to the invention is also any modificationthereof genetically designed for facilitaded purification such as e. g.an carboxyterminal polyhistidine extension.

[0014] Another embodiment of the invention is a simple process to purifyGspS to an extent that its activity as well as the inhibition thereofcan be conveniently but specifically tested by liberation of inorganicphosphate from adenosyltriphosphate (ATP) in the presence of spermidine,glutathione and magnesium ions.

[0015] The process of the invention is characterized by making use ofaqueous two phase systems containing polyethyleneglycol.

[0016] Another embodiment of the invention is the specific determinationof GspS activity by means of the detection of inorganic phosphate fromATP by partially purified GspS.

[0017] This analytical process is characterized by not being disturbedby any other ATP hydrolyzing activity enhanced by glutathione andspermidine.

[0018] Finally, another embodiment of the invention concerns apharmaceutical preparation having trypanocidal activity and comprisingan inhibitory substance according to the invention or of a protein whichcan be obtained according to the process according to the invention toproduce said protein.

[0019] The pharmaceutical composition according to the invention can becharacterized in that it can be obtained by use according to theinvention and/or by using a test system according to the invention.

[0020] The invention is now described by means of figures and examples

[0021]FIG. 1. Partial sequences of GspS from C. fasciculata compared toGsp synthetase/amidas from Escherichia coli. The amino acid numberingcorresponds to the E. coli sequence (23). *=identical amino acids,+=similar amino acids found in GspS from E. coli.

[0022]FIG. 2. Partial DNA sequence encoding GspS of C. fasciculata.Corresponding peptide sequences in the deduced amino acid sequence,which are verified by peptide sequencing of authentic GspS from C.fasciculata are underlined.

[0023]FIG. 3. Extraction of glutathionylspermidine synthetase in aqueoustwo-phase systems. GspS=glutathionylspermidine synthetase,TS=trypanothione synthetase. For extraction of GspS aqueous two-phasesystems were prepared by weighing in concentrated solutions of the phasecomponents and finally the crude extract. A poly(ethylene glycol)(PEG)/phosphate system containing 7.5% (w/w) PEG₆₀₀₀, 13% (w/w)Na-K-phosphate, pH 7.0, and 40% crude homogenate. The mixture was gentlyshaken for 10 min at room temperature and separated by centrifugation at5000 g.

[0024] With the first system we yielded an extraction of GspS into thetop phase with a purification factor of 30 in one step.

[0025] 1. System: GspS was extracted into the top phase of an aqueoustwo-phase system containing 7.5% (w/w) PEG₆₀₀₀ and 13% (w/w)Na-K-phosphate, pH 7.0. 2. System: the PEG-rich top phase of the firstsystem containing GspS was applied to a bottom phase of an identicalsystem containing water instead of cell lysate. 3. System: the top phaseof the second phase system was added to an acidified bottom phase of ablank system. The GspS was now extracted into the phosphate-rich bottomphase.

[0026]FIG. 4. SDS-PAGE analysis of the glutathionylspermidine synthetasefractions during purification. Lanes are as follows: 1, SDS markerproteins; 2, pooled fractions after chromatography on Mono P; 3, pooledfractions after chromatography on Poros 20 PE; 4, pooled fractions afterchromatography on Poros 20 Pi; 5, pooled fractions after chromatographyon Resource Q.

[0027]FIG. 5. Titration curve analysis of the homogeneousglutathionylspermidine synthetase. First dimension: isoelectricfocusing, run without protein sample; second dimension: native PAGE.

[0028]FIG. 6. Scheme of the glutathionylspermidine synthetase as a rapidequilibrium random terreactant system.

[0029]FIG. 7. pH optimum of glutathionylspermidine synthetase. Productformation of Gsp was analyzed as described under ExperimentalProcedures. Values are means±standard deviations from two independentmeasurements done at 10 and 20 min.

[0030]FIG. 8. ³¹P NMR spectra during the reaction of theglutathionylspermidine synthetase with their substrates. (A) GspS in thepresence of ATP, incubation time=0 min, scanning time=7 min; (B) GspS inthe presence of ATP, incubation time=5 h, scanning time=30 min; (C)addition of the second substrate (GSH or spermidine), incubation time=5h, scanning time=30 min; (D) addition of the third substrate (GSH orspermidine), incubation time=5 h, scanning time=30 min. The peaks areassigned to (1) inorganic phosphate, (2) ATP, γ-P, (3) ADP, β-P, (4)ADP, α-P, (5) ATP, α-P, and (6) ATP, β-P.

[0031]FIG. 9. Malachite green colorimetric assay for liberation ofinorganic phosphate. The malachite green calorimetric assay forliberation of inorganic phosphate (1) was used for fast detection ofGspS activity.

[0032] The standard assay was carried out at 25.0° C. in a volume of0.15 ml containing 50 mM Bis-Tris-propane, 50 mM Tris, pH 7.5, 5 mMMgSO_(4,) 1 mM EDTA, 5 mM DTT, 5 mM ATP, 10 mM GSH, 10 mM spermidine,and GspS. After an incubation of 10 min the malachite green reagent wasadded for detection of GspS activity.

[0033] High enzymatic GspS activity is visualized by a dark green colorof the assay.

[0034] Blank=standard assay with water instead of GspS; no liberation ofphosphate, therefore the color of the assay is yellow instead of green.

[0035] ADP significantly inhibits GspS. The type of inhibition iscompetitive with respect to ATP.

[0036] Concentration of ATP (see heading) and ADP (see table). ATP[mM]:-> 0.2 mM 0.2 mM 0.5 mM 0.5 mM 1 mM 1 mM 2 mM 2 mM 5 mM 5 mM 1 2 34 5 6 7 8 9 10 11 12 A blank 10 10 10 10 10 10 10 10 10 10 B blank 5 5 55 5 5 5 5 5 5 C blank 2 2 2 2 2 2 2 2 2 2 D blank 1 1 1 1 1 1 1 1 1 1 E0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 F 0.2 0.2 0.2 0.2 0.2 0.2 0.20.2 0.2 0.2 G 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 H 0 0 0 0 0 0 0 00 0

EXAMPLES Example 1 Purification of GspS

[0037] Production of starting material: C. fasciculata was grown in amedium previously described (27) in a 100-1 fermenter at 27° C. withcontinuous stirring (200 rpm) and aeration (0.1 vvm). Organisms wereharvested in the late logarithmic growth phase by continous flowcentrifugation. The pellet was resuspended with 100 mM HEPES buffer (pH7.5) containing 1 mM DTT and 1 mM MgSO₄. After centrifugation the cellswere stored at −20° C. GspS, Assay: The assays were carried out at 25.0°C. in a volume of 0.9 ml containing 50 mM Bis-Tris-propane, 50 mM Tris,pH 7.5, 5 mM MgSO₄, 1 mM EDTA, 5 mM DTT, 5 mM ATP, 10 mM GSH, and 10 mMspermidine (1). The assay for TS was carried out as described by Smithet al. (1). Aliquots were taken after 20 min. For thiol analysis aprecolumn derivatization with the fluorescent thiol-specific reagent,monobromobimane (Calbiochem), was used as described previously (2). Allsamples for HPLC analysis were diluted four-fold with water. Separationand analytical conditions were as described previously (28).HPLC-analysis was performed with a Jasco-HPLC-system consisting of anautosampler (851-AS), a pump (PU-980), a ternary gradient unit(LG-980-02), and a highly sensitive fluorescence detector (FP-920),which enabled a precise analysis of the small product peak in thepresence of numerous other and larger ones. An external standard (0.04mM Gsp) was used for integration calibration of the samples.

[0038] Extraction in Aqueous Two-Phase Systems: 250 g cells weresuspended in 250 ml of 20 mM Bis-Tris-propane puffer, pH 7.5, disruptedby freezing in liquid nitrogen and thawing. The crude homogenate wassubjected to an aqueous two-phase extraction at room temperature. Allother operations were performed at 4° C.

[0039] For extraction of GspS aqueous two-phase systems (total mass 900g) were prepared by weighing in concentrated solutions of the phasecomponents and finally the crude extract (FIG. 3). A poly(ethyleneglycol) (PEG)/phosphate system containing 7.5% (w/w) PEG₆₀₀₀, 13% (w/w)Na—K-phosphate, pH 7.0, and 40% crude homogenate (or water in the blanksystems) was used. The mixture was gently shaken for 10 min at roomtemperature and separated by centrifugation at 5000 g. The top phase wassucked off and applied to a bottom phase of a blank system. Aftermixing, centrifugation, and separation of the phases the PEG-rich topphase of the second phase extraction was mixed with a blank bottomphase, adjusted to pH 6.0 with HCl. This third system was mixed again,centrifuged, and separated. Now the GspS was found in the phosphate-richbottom phase.

[0040] Diafiltration: The phosphate-rich third bottom phase and otherpooled enzyme fractions were diafiltrated with an omega membrane with acut-off of 30 kDa (Filtron Minisette) using a Pro Flux M12 (Amicon) at0.2 MPa and a 500-fold volume of 2 mM Bis-Tris-propane buffer, pH 8.0.

[0041] Resource Q Chromatography: A BioLogic-System (Bio-Rad) was usedat 4° C. for all chromatographies. The diafiltrated protein mixture wasapplied onto a Resource Q column (6 ml) (Pharmacia) equilibrated with 2mM Bis-Tris-propane buffer, pH 8.0. After washing with 10 column volumesof equilibration buffer, the bound proteins were eluted at a flow rateof 1 ml/min with a gradient of 0.0 to 0.4 M KCl (100% B) as follows: t=0min, B=0%; t=20 min, B=15%; t=40 min, B=15%; t=60 min, B=30%; t=120 min,B=30%; t=150 min, B=100%. The GspS eluted at 0.27 M KCl and the pooledactive fractions were diafiltrated with 2 mM Bis-Tris-propane buffer, pH6.0.

[0042] Poros 20 Pi Chromatography: The diafiltrated proteins wereapplied onto Poros 20 Pi (0.46×10 cm, 1.7 ml) (Perseptive Bioystems)equilibrated with 2 mM Bis-Tris-propane buffer, pH 6.0. After washingwith 10 column volumes of equilibration buffer, bound proteins wereeluted at a flow rate of 4 ml/min with a gradient of 0 to 1 M NaCl (100%B) as follows: t=0 min, B=0%; t=8 min, B=35%; t=16 min, B=35%; t=17 min,B=37%; t=21 min B =37%; t=25 min, B=100%. GspS eluted at 0.7 M NaCl.

[0043] Poros 20 PE Chromatography: Pooled active fractions were adjustedto 1 M ammonium sulfate and applied onto a hydrophobic interactionchromatography column Poros 20 PE (0.46×10 cm, 1.7 ml) (PerseptiveBiosystems) equilibrated with 20 mM Bis-Tris-propane buffer, pH 8.0,containing 1 M ammonium sulfate, washed with 10 column volumes ofequilibration buffer, and eluted with a linear gradient of 1 to 0 Mammonium sulfate and a flow rate of 4 ml/min over 7.5 min. GspS elutedat 0.75 M ammonium sulfate. Pooled active fractions were diafiltratedwith 10 mM Bis-Tris-propane buffer, pH 6.8.

[0044] Mono P Chromatography: The diafiltrated fraction was applied ontoa Mono P HR 5/20 column (4 ml) (Pharmacia) for anion exchangechromatography. The column was equilibrated with 10 mM Bis-Tris-propanebuffer, pH 6.8. After washing with 10 column volumes of equilibrationbuffer, bound proteins were eluted with a gradient of 0 to 1 M NaCl(100% B) as follows: t=0 min, B=0%; t=20 min, B=25%; t=40 min, B=25%;t=60 min, B=50%; t=80 min, B=50%; t=100 min, B=100%. The flow rate was 1ml/min. GspS eluted at 0.45 M NaCl.

[0045] Results: The purification strategy outlined above resulted in aGspS preparation with a specific activity of 37.6 U/mg at an overallyield of about 20%. The purification factor achieved was 12,500. As isseen from table 1, the phase distribution system applied proved to behighly efficient in enriching GspS.

[0046] The optimized procedure was based on a factorial design of phasecompositions (31), i.e. PEG₆₀₀₀/phosphate (7.5/13% (w/w)),PEG₄₀₀₀/phosphate (8/14% (w/w)), PEG₁₅₅₀/phosphate (9/18% (w/w)), eachtested at pH 4.0, 5.5, and 7.0 and containing 40% cell lysate. Bycentrifugation the cell debris were concentrated in a gum-likeinterphase if the pH of the system was 5.5. A graphical evaluation ofthe experimental data (not shown) clearly demonstrated a significantincrease in the partition coefficient of GspS with increasing pH, and adecrease in the partition coefficient of the total protein withincreasing molecular weight of PEG. The best system, containing 7.5%(w/w) PEG₆₀₀₀, 13% (w/w) phosphate, pH 7.0, yielded an extraction ofGspS into the top phase (FIG. 3) with a purification factor of 30 in onestep.

[0047] Some residual turbidity left in the top phase of the initialextraction could be eliminated by a second extraction step, mixing theprimary top phase with a bottom phase of an identical blank system. Bythese systems a proteolytic activity, as observed with casein yellow,and an ATPase activity were quantitatively removed by extraction intothe bottom phases. Simultaneously GspS was completely separated fromtrypanothione synthetase (TS) activity. While GspS was recoveredcompletely in the top phase, TS activity was extracted into the bottomphase (FIG. 3), but proved to be unstable and was not purified further.After two a extractions into top phases GspS was essentially free ofinterfering enzymatic activities and could be precisely quantitated byATP hydrolysis in the presence of glutathione and spermidine. The finalchromatographic purification of GspS, however, was impaired by the highphosphate concentration and viscosity of the top phase in which theenzyme was dissolved. GspS was therefore extracted from the second topphase into the bottom phase of a third system by lowering its pH to 6.0without loss of activity. The GspS in the phosphate-rich bottom phasewas diafiltrated and then could be loaded onto a Resource Q column. GspSthus purified appeared homogeneous by SDS-PAGE (FIG. 4) and by titrationcurve analysis (FIG. 5).

Example 2 Determination of Physical Parameters of GspS

[0048] Molecular mass estimation by gel permeation chromatography:Proteins were applied onto a gel permeation chromatography columnSuperose 12 (HR 10/30) (Pharmacia) equilibrated with 20 mMBis-Tris-propane buffer, pH 7.5 containing 0.15 M NaCl and eluted with aflow rate of 0.3 ml/min. Blue dextran (2,000 kDa), thyroglobulin (669kDa), ferritin (440 kDa), β-amylase (200 kDa), alcohol dehydrogenase(150 kDa), bovine serum albumin (67 kDa), and carbonic anhydrase (30kDa) were used as standards.

[0049] Gel permeation chromatography on Superose 12 indicated amolecular mass of b 79 l kDa. A small activity peak eluted at about 170kDa suggesting a slight tendency of the enzyme to dimerize. In essence,however, GspS of C. fasciculata was present as a monomeric enzyme of 79kDa.

[0050] Electrophoresis: The subunit molecular weight was determined bySDS-PAGE (28) using a PhastGel Gradient 8-25 (Pharmacia) with thefollowing molecular weight standards: phosphorylase b (94 kDa), bovineserum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa),trypsin inhibitor (20.1 kDa), and α-lactalbumin (14.4 kDa). A subunitmolecular mass of GspS of 78 kDa was estimated by SDS-PAGE.

[0051] The native molecular weight was determined by native PAGE using aPhastGel Gradient 8-25 (Pharmacia) with the same following molecularweight standards and additionally: thyroglobulin (669 kDa), ferritin(440 kDa), catalase (232 kDa), and lactate dehydrogenase (140 kDa).

[0052] A molecular weight of 78 kDa was obtained by gradient gelelectrophoresis of the native enzyme. The identity of the 78 kDa bandwith GspS was confirmed by activity staining, i.e. phosphate liberationupon incubation with Mg²⁺-ATP, GSH, and spermidine (not shown). Theisoelectric point was determined by isoelectric focusing using aPhastGel IEF 3-9 (Pharmacia) with a broad pI calibration kit and bytitration curve analysis with PhastGel IEF 3-9. The latter technique isa two-dimensional electrophoresis. In the first dimension, a pH gradientis generated. The gel is then rotated clockwise 90° and the sample isapplied perpendicular to the pH gradient across the middle of the gel(29).

[0053] For detection of GspS activity after isoelectric focusing thegels were cut into two pieces, one was silver stained for proteindetection and the second was incubated for 15 min at room temperature ina solution of 100 mM HEPES, pH 7.0, 5 mM MgSO₄, 1 mM EDTA, 5 mM DTT, 10mM GSH, 10 mM spermidine, and 2 mM ATP. After 15 min 2.5 ml of astaining solution containing malachite green, ammonium molybdate andTween 20 (1) was added. Lanes containing active GspS showed a dark greencolour after few minutes. The isoelectric point deduced from isoelectricfocusing was at pH 4.6.

Example b 3 Amino Acid Sequencing

[0054] SDS-PAGE of purified GspS was performed at a constant current of20 mA in a separating gel (T=7.5%). For blotting, the proteins weretransferred for 1.5 h onto a PVDF-membrane at 40 V/70 mA in a buffercontaining 25 mM Tris base, 192 mM glycine, and 10% (v/v) methanol. Theblot was stained with Coomassie blue.

[0055] For peptide sequencing the band corresponding to a molecular massof 78 kDa was cut out. This material was washed and digested withendoproteinase Lys-C as described before (30) and separated byreversed-phase HPLC (30). Peptide peaks were detected at 214 nm andcollected manually. Aliquots of 15-30 μl were applied directly tobiobrene-coated, precycled glass fibre filters of an sequencer (AppliedBiosystems 470A) sequencer with standard gas-phase programs of themanufacturer.

[0056] N-terminal amino acid sequencing proved unsuccessful obviouslydue to a N-terminal blocking group. After proteolytic cleavage withendoproteinase Lys-C, however, a total of 11 peptides could be recoveredfrom HPLC in a quality to allow sequencing. Out of these peptides, 7could unambigously be aligned to the deduced GspS sequence of E. colirecently published by Bollinger et al. (25) (FIG. 1). GspS of E. coliand of C. fasciculata thus appeared to be phylogenetically related. Butbased on the limited sequence information, the sequence similaritybetween these enzymes, with only 40% identities, appears rather low.

Example b 4 Kinetic analysis

[0057] All kinetic experiments were carried out at 25.0° C. in a volumeof 0.9 ml containing 50 mM Bis-Tris-propane, 50 mM Tris, pH 7.5, 1 mMEDTA, 5 mM DTT and variable concentrations of ATP (0.10, 0.13, 0.18,0.28, 0.66 mM ATP), GSH (0.36, 0.47, 0.66, 1.11, 3.57 mM GSH), andspermidine (0.36, 0.47, 0.66, 1.11, 3.57 mM spermidine), respectively.The enzymatic tests for kinetic studies except the ADP inhibitionstudies were performed in the presence of phosphoenolpyruvate (10 mM)and pyruvate kinase (0.5 units). A fixed magnesium concentration of 5 mMand a GspS content of 0.072 mg (0.923 μM) was used. Aliquots were takenat 15 min and 30 min. GspS activity was analyzed by productdetermination as described in example 1.

[0058] The experimental data thus obtained classify the kineticmechanism of GspS as an equilibrium random-order mechanism. Whether thecomplexation of the individual substrates occurs absolutelyindependently of each other or whether the binding substrates mutuallyaffect affinities of cosubstrates, is less easily decided. The apparentK_(m) values for the different substrates, however, are notsignificantly affected by the concentrations of the respectivecosubstrates. Consequently, the deduced dissociation constants of thecorresponding binary, ternary and quarternary complexes are very closefor a given substrate and not significantly different. This would indeedimply a mutually independent random addition of substrates. But withregard to the inevitable scatter of data it can not be exluded that someroute leading to the quarternary complex is slightly favoured. However,a rapid equilibrium random-order mechanism, as depicted in FIG. 6,complies best with the experimental data. Based on this assumption, thelimiting K_(m) values are defined as the dissociation constants of thequarternary complexes, numerically 0.25±0.02, 2.51±0.33, and 0.47±0.09mM for Mg²⁺-ATP, GSH, and spermidine, respectively. The rate limitingvelocity constant then can be calculated to be 415±78 min⁻¹.

[0059] A quarternary complex mechanism implies that all three substratesmust be assembled at the enzyme before a reaction can proceed. In orderto check this hypothesis, we subjected the enzyme to long-term exposurewith Mg²⁺-ATP plus one of the additional substrates and monitored apotential partial reaction by ³¹P NMR.

[0060]³¹P NMR spectra were recorded on a Bruker ARX 400 NMRspectrometer, at 162 MHz and locked to the deuterium resonance of D₂O,to detect potential partial reactions.

[0061] The experiments were carried out at 25.0° C. in a volume of 0.6ml containing 50 mM Bis-Tris-propane, 50 mM Tris, pH 7.5, 5 mM MgSO₄, 1mM EDTA, 5 mM DTT, in the presence of 20% D₂O. Spectra were recorded atthe beginning of the experiment and after the addition of the substrates(5 mM ATP, 10 mM GSH, and 10 mM spermidine).

[0062]FIG. 8 demonstrates that with all combinations of substrates noATP turnover could be observed within 5 hours unless the third substratewas added. These findings strongly support the assumption of aquarternary complex mechanism and explain the absence of any ATPaseactivity of GspS. Neither can the presumed catalytic intermediateglutathionylphosphate be formed in any detectable amount by anincomplete catalytic complex.

[0063] As already mentioned ADP significantly inhibits GspS whichrenders it difficult to measure GspS activity in the absence of an ATPregenerating system. The type of inhibition is competitive with respectto ATP. A K_(i) of 80 μ M was calculated which is in the range ofphysiological ADP concentrations. GspS also proved to be feed-backinhibited by TSH with a K_(i) of 480 μM, which is competed by GSH.

Example b 5 pH-Optimum of GspS

[0064] The activity of GspS was measured essentially as described inexample 4 but at pH values ranging from 6 -8. GspS shows a flat pHoptimum near pH 7.5 (FIG. 7).

Example 6 Use of Malachite Green Colorimetric Assay for Liberation ofInorganic Phosphate in GspS Preparations Partially Purified According toExample b 1

[0065] The liberation of inorganic phosphate from ATP can be easilyvisualized by malachite green (FIG. 9). The test is amply used tomonitor ATP hydrolyzing activities and is correspondingly unspecific.Surprisingly, the GspS preparation after aqueous two phase extraction,as described in example 1, proved to be free of any significantspontaneous ATP-hydrolyzing activity. This finding enabled us to usethis fast and convenient test to measure specifically GspS activitywhich is accompanied by release of inorganic phosphate from ATP in thepresence of glutathione, spermidine, and magnesium ions. The test can beeasily adapted to mass screening as outlined below.

[0066] The malachite green calorimetric assay for liberation ofinorganic phosphate (1l) was used for fast detection of GspS activityduring purification after column chromatography and for GspSlocalization on gels.

[0067] The disclosure comprises also that of EP 96 120 014.4 filed Dec.12, 1996, the entire disclosure of which is incorporated herein byreference.

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1 9 20 amino acids amino acid single linear protein NO NO N-terminal 1Val Pro Phe Gly Glu Val Gln Gly Tyr Ala Pro Gly His Ile Pro Ala 1 5 1015 Tyr Ser Asn Lys 20 13 amino acids amino acid single linear protein NONO N-terminal 2 Ser Ile Ile Thr Gly Leu Asp Ser Pro Phe Ala Ala Ile 1 510 6 amino acids amino acid single linear protein NO NO N-terminal 3 ThrTyr Glu Pro Thr Glu 1 5 10 amino acids amino acid single linear proteinNO NO N-terminal 4 Asn Glu Ile Pro Arg Pro Leu Thr His Lys 1 5 10 8amino acids amino acid single linear protein NO NO N-terminal 5 Leu AspLeu Asn Asp Pro Ala Glu 1 5 19 amino acids amino acid single linearprotein NO NO N-terminal 6 Ile Leu Pro Ile Ile Tyr His Asn His Pro AspHis Pro Ala Ile Leu 1 5 10 15 Arg Ala Glu 14 amino acids amino acidsingle linear protein NO NO N-terminal 7 Ile Val Gly Arg Val Gly Arg AsnVal Thr Ile Thr Asp Gly 1 5 10 573 amino acids amino acid single linearprotein NO NO Modified-site 191 /note= “Xaa = Lys or Asn” Modified-site463 /note= “Xaa = Val or Asp” Modified-site 479 /note= “Xaa = Val orGly” 8 Tyr Ser Asn Lys His Asp His Phe Phe Ser Gly Glu Arg Ser Ile Asp 15 10 15 Asp Asn Val Phe Cys Gly Phe Lys Tyr Gln Cys Val Glu Phe Ala Arg20 25 30 Arg Trp Leu Leu Glu Arg Lys Gly Leu Val Leu Pro Asp Val Asn Trp35 40 45 Ala Cys His Ile Phe Lys Leu Lys Ser Val Lys Asp Ala Ala Thr Ala50 55 60 Glu Glu Val Pro Val Ile Ala Val Arg Asn Gly Thr Glu Ala Lys Pro65 70 75 80 Glu Pro Asp Thr Leu Ile Ile Tyr Pro Ser Ser Asp Val Asn ThrVal 85 90 95 Gly His Val Gly Ala Ile Thr Glu Val Gly Asp Asp Tyr Val CysIle 100 105 110 Ala Asp Gln Asn Tyr Arg Phe His Lys Trp Glu Ala Ser TyrSer Tyr 115 120 125 Lys Leu Lys Leu Gln His Lys Asp Gly Val Trp Thr IleIle Asp Asp 130 135 140 Ile Asp Pro Asn Asp Val Glu Ile Pro Leu Gly TrpVal Thr Phe Pro 145 150 155 160 Gly Tyr Glu Asn Arg Pro Glu Gly Ala AlaPro Pro Ala Leu His Pro 165 170 175 Ser Leu His Phe Gln Pro Pro Glu GluPro Tyr Leu Val Arg Xaa Thr 180 185 190 Tyr Glu Pro Thr Glu Thr Lys AlaAsn Trp Leu Asp Leu Asn Asp Pro 195 200 205 Ala Glu Lys Leu Phe Val GluGlu Phe Gly Met Asp Val Ser Arg Ser 210 215 220 Arg Leu Glu Glu Thr ThrVal Asn Tyr Tyr Glu Cys Asp His Glu Phe 225 230 235 240 His Leu Arg CysIle Ala Tyr Gly Thr Gln Leu His Asp Tyr Phe Met 245 250 255 Glu Ala ThrAla Gln Val Ile Asn Asp Glu Arg Leu Arg Ile Phe Lys 260 265 270 Ile ProGlu Glu Leu Trp Pro Arg Met Arg His Ser Trp Lys Tyr Gln 275 280 285 GlnThr Tyr Ile Ser Gly Arg Phe Asp Phe Ala Tyr Asn Asn Glu Thr 290 295 300His Gln Met Lys Cys Phe Glu Tyr Asn Ala Asp Ser Ala Ser Thr Leu 305 310315 320 Leu Glu Cys Gly Arg Ile Gln Gln Lys Trp Ala Glu Ser Ala Gly Leu325 330 335 Asp Lys Glu Gly Thr Arg Gly Ser Gly Trp Ala Val Glu Arg AsnLeu 340 345 350 Pro Thr Ala Trp Ala Thr Cys Gly Ala Thr Gly Arg Val HisPhe Leu 355 360 365 Val Asp Asp Glu Lys Glu Glu Gln Tyr Thr Ala Leu TyrCys Leu Gln 370 375 380 Ala Arg Lys Arg Gly Leu Glu Gly Lys Leu Cys ValMet Tyr Asp Glu 385 390 395 400 Phe Arg Phe Asn Glu Glu Gly Tyr Val ValAsp Ser Asp Gly Val Arg 405 410 415 Val Arg Asn Ile Trp Lys Thr Trp MetTrp Glu Ser Ala Ile Ser Asp 420 425 430 Tyr Phe Ala Ala Gln Ala Glu ArgVal Arg Leu Glu Gly Asp Ala Ala 435 440 445 Asp Lys Val Arg Leu Cys AspLeu Met Leu Gly Lys Asp Trp Xaa Ile 450 455 460 Leu Tyr Phe Glu Pro MetTrp Lys Leu Ile Pro Ser Asn Lys Xaa Ile 465 470 475 480 Leu Pro Ile IleTyr His Asn His Pro Asp His Pro Ala Ile Leu Arg 485 490 495 Ala Glu TyrGlu Leu Thr Asp Glu Leu Leu Arg Cys Gly Tyr Ala Arg 500 505 510 Lys ProIle Val Cys Arg Val Gly Arg Asn Val Thr Ile Thr Asp Gly 515 520 525 ThrGly Glu Val His Ala Glu Ser Gly Gly Asn Phe Gly Glu Arg Asp 530 535 540Met Ile Tyr Gln Glu Leu Phe Ser Leu Thr Lys Gln Asp Gly Tyr Tyr 545 550555 560 Ala Ile Ile Gly Gly Met Leu Gly Asp Ala Phe Ser Gly 565 570 1719base pairs nucleic acid single linear DNA (genomic) NO NO misc_feature483 /note= “M = C or A” misc_feature 489 /note= “R = G or A”misc_feature 546 /note= “S = C or G” misc_feature 555 /note= “R = G orA” misc_feature 573 /note= “K = G or T” misc_feature 624 /note= “Y = Cor T” misc_feature 627 /note= “W = A or T” misc_feature 687 /note= “S =G or C” misc_feature 933 /note= “R = G or A” misc_feature 954 /note= “R= G or A” misc_feature 966 /note= “R = G or A” misc_feature 981 /note=“S = G or C” misc_feature 984 /note= “R = G or A” misc_feature 1388/note= “W = T or A” misc_feature 1436 /note= “K = T or G” 9 TACAGCAACAAGCACGATCA CTTCTTCTCG GGTGAGCGCA GCATTGACGA TAACGTCTTC 60 TGCGGCTTCAAGTACCAGTG CGTCGAGTTC GCGCGCCGCT GGCTGTTGGA GCGGAAGGGG 120 CTGGTGCTGCCGGACGTGAA TTGGGCGTGC CACATCTTCA AGCTCAAGAG CGTGAAGGAT 180 GCCGCGACGGCGGAGGAGGT GCCGGTGATC GCCGTGCGCA ACGGCACGGA GGCGAAGCCG 240 GAGCCCGACACGCTGATCAT CTACCCCTCG TCGGACGTCA ACACCGTGGG CCACGTCGGC 300 GCCATCACGGAGGTCGGCGA CGACTACGTG TGCATTGCGG ACCAGAACTA CCGCTTTCAC 360 AAGTGGGAGGCGTCCTACTC CTACAAGTTG AAGCTGCAGC ACAAGGATGG GGTTTGGACG 420 ATCATCGACGACATCGACCC CAACGATGTC GAGATTCCGC TTGGCTGGGT GACCTTCCCC 480 GGMTACGARAACCGGCCGGA AGGCGCCGCG CCACCGGCGC TGCACCCCTC TCTCCACTTC 540 CAGCCSCCGGAGGARCCGTA CCTGGTCCGC AAKACGTACG AGCCGACGGA GACGAAGGCG 600 AACTGGCTGGATTTGAACGA CCCYGCWGAG AAGCTCTTTG TGGAGGAGTT CGGCATGGAC 660 GTCAGCCGCTCCCGCCTCGA GGAGACSACG GTGAACTACT ACGAGTGCGA CCATGAGTTC 720 CACCTCCGCTGCATCGCCTA CGGGACGCAG CTGCACGACT ACTTCATGGA GGCCACCGCG 780 CAGGTCATCAACGACGAGCG GCTCCGCATC TTTAAGATTC CAGAGGAGCT GTGGCCCCGC 840 ATGCGCCACTCCTGGAAGTA CCAGCAGACG TACATCTCTG GCCGCTTTGA CTTCGCCTAC 900 AACAACGAGACGCACCAGAT GAAGTGCTTC GARTACAACG CCGACAGCGC GTCRACGCTG 960 CTGGARTGCGGCCGCATTCA SCARAAGTGG GCCGAGTCGG CGGGGCTGGA CAAGGAGGGC 1020 ACGCGCGGCTCCGGCTGGGC CGTCGAGCGC AACCTGCCGA CCGCGTGGGC CACCTGCGGC 1080 GCCACTGGTCGCGTGCACTT CCTCGTGGAC GATGAGAAGG AGGAGCAGTA CACGGCCCTT 1140 TACTGCCTGCAGGCGCGGAA GCGTGGGCTG GAGGGGAAGC TGTGCGTCAT GTACGACGAG 1200 TTCCGCTTCAACGAGGAGGG CTACGTCGTG GACAGCGATG GGGTGCGGGT GCGCAACATT 1260 TGGAAGACGTGGATGTGGGA GTCGGCCATC AGCGACTACT TCGCCGCGCA GGCCGAGCGC 1320 GTGCGACTGGAAGGCGACGC CGCCGACAAG GTGCGGCTCT GTGACCTGAT GCTCGGCAAG 1380 GACTGGGWCATCTTGTACTT TGAGCCGATG TGGAAGCTGA TCCCGAGCAA CAAGGKCATC 1440 CTGCCCATCATCTACCACAA CCACCCTGAT CACCCGGCGA TCCTGCGCGC TGAGTACGAG 1500 CTCACCGACGAGCTCCTACG CTGTGGCTAC GCCAGGAAGC CGATTGTTTG CCGTGTCGGC 1560 CGCAACGTCACCATCACCGA CGGCACGGGT GAGGTGCACG CCGAGTCGGG CGGCAACTTC 1620 GGCGAGCGGGATATGATTTA CCAGGAGCTC TTCTCCCTGA CGAAGCAGGA TGGTTATTAC 1680 GCGATCATCGGCGGCATGCT GGGCGACGCG TTCAGCGGC 1719

We claim:
 1. A protein catalyzing the synthesis ofglutathionylspermidine having a pH optimum of said synthesis of about pH7.5 and having a molecular weight of 78,000±3,000 Da.
 2. A proteinaccording to claim 1 comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4,SEQ ID NO 5, SEQ ID NO 6, and SEQ ID NO 7 as shown in FIG.
 1. 3. Aprotein according to claim 1 isolated from a species of the family oftrypanosomatids (Trypanosomatidae).
 4. A protein according to claim 3wherein said species is selected from the group consisting ofTrypanosoma sp., Leishmania sp., Herpetomonas sp., Leptomonas sp.,Blastocrithidia sp., Crithidia sp., and Phytomotnas sp.
 5. A proteinaccording to claim 4 wherein said species is C. fasciculata.
 6. Aprotein according to claim 1 produced by the method comprising the stepsof: (a) culturing a host cell transformed with DNA selected from DNAencoding any one of the amino acid sequences SEQ ID NO 1, SEQ ID NO 2,SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, and SEQ ID NO 7 asshown in FIG. 1 and DNA encoding the nucleotide sequence SEQ ID No 8shown in FIG. 2, and, (b) isolating said protein from said host cell orits growth medium.
 7. A protein according to claim 1 comprising apartial deduced amino acid sequence selected from the group consistingof SEQ ID NO 8 as shown in FIG. 2, and sequences homologous to said SEQID NO 8 having the same number of amino acids as SEQ ID NO 8 and beingidentical to SEQ ID NO 8 in more than 70% of the amino acid residues. 8.A protein according to claim 7 wherein said sequence is identical to SEQID NO 8 in more than 75% of the amino acid residues.
 9. A proteinaccording to claim 1 encoded in part by a partial DNA sequence selectedfrom the group consisting of SEQ ID NO 9 shown in FIG. 2 and other DNAsequences having the same number of nucleotides and being identical toSEQ ID NO 9 in more than 70% of the nucleotides.
 10. A protein accordingto claim 9 wherein said other DNA sequence is identical to SEQ ID NO 9in more than 75% of the nucleotides.
 11. A protein according to claim 9wherein the complementary strand of said other DNA sequence hybridizesto SEQ ID NO 9 at a temperature of at least 25° C. and at a NaClconcentration of 1M.
 12. A process for recovering a protein according toclaim 1 comprising the steps of (a) homogenizing cells belonging toTrypanosomatidae; (b) extracting the resulting homogenate with anaqueous multi-phase system; (c) separating the resulting phasecontaining the protein; and (d) optionally, isolating the protein fromsaid phase of (c).
 13. A process according to claim 12 wherein theextraction step (b) is carried out with an aqueous two-phase system. 14.A process according to claim 12 wherein one of the aqueous phases of themulti-phase system contains at least one organic polymer, and anotherphase of the multi-phase system is an aqueous solution of at least onesalt.
 15. A process according to claim 14 wherein said organic polymercomprises polyethylene glycol (PEG) having a molecular weight of morethan 1500 Da.
 16. A process according to claim 15 wherein said PEG has amolecular weight more than 6000 Da.
 17. A process according to claim 14wherein said one aqueous phase contains PEG having a molecular weight ofabout 6000 Da, and the other aqueous phase is a phosphate solution. 18.A process according to claim 17 wherein said phosphate solution has a pHof about
 7. 19. A process according to claim 7 comprising the step,before step (d) of extracting the protein contained in said phaseseparated in step (c) by means of another aqueous phase optionallyfollowed by at least one additional step of extracting the protein fromthe phase with an aqueous phase up to substantially complete separationof enzyme activities cleaving ATP, other than the protein wanted.
 20. Aprocess according to claim 19 wherein the other aqueous phase is free ofpolyethylene glycol (PEG) or has a lower PEG concentration than anyphase of the multi-phase system of step (b).
 21. A process according toclaim 19 comprising the step of extracting the phase separated in step(c) by lowering the pH thereof.
 22. A process according to claim 21wherein said pH is lowered to pH 6 or below.
 23. A process according toclaim 19 wherein said another aqueous phase contains at least oneorganic polymer or at least one dissolved salt.
 24. A process accordingto claim 7 wherein said cells are selected from the group consisting ofTrypanosoma, Leishmania, Herpetomonas, Leptomonas, Blastocrithidia,Crithidia, and Phytomotnas.
 25. A process according to claim 24 whereinsaid cells are Crithidia fasciculata cells.
 26. A process according toclaim 25 wherein said cells are a-pathogenic.
 27. A method ofidentifying compounds having trypanocidal activity (GspS inhibitors)comprising the steps of (a) contacting the protein of claim 7 with saidcompound in an aqueous phase and (b) measuring inhibition of theactivity of the protein.
 28. The method according to claim 27 whereinthe activity of the protein is measured by measuring ATP hydrolysis. 29.GspS inhibitors identified by the method of claim
 27. 30. Isolated GspSinhibitors of claim
 29. 31. Pharmaceutical composition comprising a GspSinhibitor according to claim 29 and at least one of a carrier and anadjuvant.